Vicenta Devesa

Arsenic (+3 Oxidation State) Methyltransferase and the Methylation of Arsenicals

Metabolic conversion of inorganic arsenic into methylated products is a multistep process that yields mono-, di-, and trimethylated arsenicals. In recent years, it has become apparent that formation of methylated metabolites of inorganic arsenic is not necessarily a detoxification process. Intermediates and products formed in this pathway may be more reactive and toxic than inorganic arsenic. Like all metabolic pathways, understanding the pathway for arsenic methylation involves identification of each individual step in the process and the characterization of the molecules which participate in each step. Among several arsenic methyltransferases that have been identified, arsenic (+3 oxidation state) methyltransferase is the one best characterized at the genetic and functional levels. This review focuses on phylogenetic relationships in the deuterostomal lineage for this enzyme and on the relation between genotype for arsenic (+3 oxidation state) methyltransferase and phenotype for conversion of inorganic arsenic to methylated metabolites. Two conceptual models for function of arsenic (+3 oxidation state) methyltransferase which posit different roles for cellular reductants in the conversion of inorganic arsenic to methylated metabolites are compared. Although each model accurately represents some aspects of enzyme’s role in the pathway for arsenic methylation, neither model is a fully satisfactory representation of all the steps in this metabolic pathway. Additional information on the structure and function of the enzyme will be needed to develop a more comprehensive model for this pathway.

Molecular Mechanisms of the Diabetogenic Effects of Arsenic: Inhibition of Insulin Signaling by Arsenite and Methylarsonous Acid

Background Increased prevalences of diabetes mellitus have been reported among individuals chronically exposed to inorganic arsenic (iAs). However, the mechanisms underlying the diabetogenic effects of iAs have not been characterized. We have previously shown that trivalent metabolites of iAs, arsenite (iAsIII) and methylarsonous acid (MAsIII) inhibit insulin-stimulated glucose uptake (ISGU) in 3T3-L1 adipocytes by suppressing the insulin-dependent phosphorylation of protein kinase B (PKB/Akt). Objectives Our goal was to identify the molecular mechanisms responsible for the suppression of PKB/Akt phosphorylation by iAsIII and MAsIII. Methods The effects of iAsIII and MAsIII on components of the insulin-activated signal transduction pathway that regulate PKB/Akt phosphorylation were examined in 3T3-L1 adipocytes. Results Subtoxic concentrations of iAsIII or MAsIII had little or no effect on the activity of phosphatidylinositol 3-kinase (PI-3K), which synthesizes phosphatidylinositol-3,4,5-triphosphate (PIP3), or on phosphorylation of PTEN (phosphatase and tensin homolog deleted on chromosome ten), a PIP3 phosphatase. Neither iAsIII nor MAsIII interfered with the phosphorylation of 3-phosphoinositide-dependent kinase-1 (PDK-1) located downstream from PI-3K. However, PDK-1 activity was inhibited by both iAsIII and MAsIII. Consistent with these findings, PDK-1-catalyzed phosphorylation of PKB/Akt(Thr308) and PKB/Akt activity were suppressed in exposed cells. In addition, PKB/Akt(Ser473) phosphorylation, which is catalyzed by a putative PDK-2, was also suppressed. Notably, expression of constitutively active PKB/Akt restored the normal ISGU pattern in adipocytes treated with either iAsIII or MAsIII. Conclusions These results suggest that inhibition of the PDK-1/PKB/Akt-mediated transduction step is the key mechanism for the inhibition of ISGU in adipocytes exposed to iAsIII or MAsIII, and possibly for impaired glucose tolerance associated with human exposures to iAs.

Comprehensive analysis of arsenic metabolites by pH-specific hydride generation atomic absorption spectrometry

In a variety of biological systems, inorganic arsenic (iAs) is metabolized to yield methylated arsenicals that contain arsenic in +5 or +3 oxidation states. Atomic absorption spectrometry (AAS) coupled with a pH-specific generation of arsines has been used for selective analysis of trivalent and pentavalent inorganic, mono-, and dimethylated arsenicals in biological matrices. We have optimized this method to permit simultaneous detection and quantification of all relevant metabolites of iAs, including trimethylarsine oxide (TMAsVO). The optimization includes increasing the density of the chromatographic adsorbent used for cold-trapping of generated arsines and modification of the temperature gradient for release of arsines from the cold trap. These modifications improve the boiling-point separation of arsine, methylarsine, dimethylarsine, and trimethylarsine before the detection by AAS. Arsines from trivalent arsenicals and from TMAsVO are selectively generated at pH 6. At pH 1, arsines are generated from both tri- and pentavalent arsenicals. Thus, the optimized technique permits analysis of arsenite (iAsIII), arsenate (iAsV), monomethylarsonic acid (MAsV), monomethylarsonous acid (MAsIII), dimethylarsinic acid (DMAsV), dimethylarsinous acid (DMAsIII), and TMAsVO. The detection limits range from 0.14 ng As (for TMAsVO) to 0.40 ng As (for iAsV). Calibration curves are linear over the concentration range of 0.5–100 ng As. Recoveries vary between 85 and 124%. The precision of the method in various biological matrices ranges from 1.0 to 14.5%. Using the optimized technique, both trivalent and pentavalent methylated and dimethylated arsenicals, but not TMAsVO, have been detected in cultured primary human hepatocytes exposed to iAsIII. In contrast, TMAsVO was detected as the final product of in vitro methylation of iAsIII by rat AsIII-methyltransferase, cyt19. TMAsVO was also detected in the urine of mice treated with MAsV or DMAsV. Thus, the optimized method improves the efficiency of arsenic speciation analysis in biological matrices, providing a more comprehensive picture of the role of metabolism in the disposition and action of iAs.

In Vitro Study of Transporters Involved in Intestinal Absorption of Inorganic Arsenic

Inorganic arsenic (iAs) [As(III)+As(V)] is a drinking water contaminant, and human exposure to these arsenic species has been linked with a wide range of health effects. The main path of exposure is the oral route, and the intestinal epithelium is the first physiological barrier that iAs must cross in order to be absorbed. However, there is a lack of information about intestinal iAs absorption. The aim of this study was to evaluate the participation of certain transporters [glucose transporters (GLUT and SGLT), organic anion transporting polypeptides (OATPs), aquaporins (AQPs), and phosphate transporters (NaPi and PiT)] in intestinal absorption of As(V) and As(III), using the Caco-2 cell line as a model of the intestinal epithelium. For this purpose, the effects of chemical inhibition and gene silencing of the transporters of interest on iAs uptake were evaluated, and also the differential expression of these transporters after treatment with iAs. The results show that chemical inhibition using rifamycin SV (OATP inhibitor), phloridzin (SGLT inhibitor), phloretin (GLUT and AQP inhibitor), and copper sulfate (AQP inhibitor) leads to a significant reduction in the apparent permeability and cellular retention of As(III). RT-qPCR indicates up-regulation of GLUT2, GLUT5, OATPB, AQP3, and AQP10 after exposure to As(III), while exposure to As(V) increases the expression of sodium-dependent phosphate transporters, especially NaPiIIb. Gene silencing of OATPB, AQP10, and GLUT5 for As(III) and NaPiIIb for As(V) significantly reduces uptake of the inorganic forms. These results indicate that these transporters may be involved in intestinal absorption of iAs.

Characterization of the Intestinal Absorption of Arsenate, Monomethylarsonic Acid, and Dimethylarsinic Acid Using the Caco-2 Cell Line

Many toxicological studies have been conducted with arsenic species in target organ cell lines. However, although epithelial gastrointestinal cells constitute the first barrier to the absorption of contaminants, studies using intestinal cells are scarce. The present study examines absorption through the intestinal epithelium of the pentavalent arsenic species most commonly found in foods [arsenate, As(V); monomethylarsonic acid, MMA(V); and dimethylarsinic acid, DMA(V)], using the Caco-2 cell line as a model. Different concentrations (1.3-667.6 microM) and culture conditions (media, pH, addition of phosphates, and treatment with ethylenediaminetetraacetic acid) were evaluated to characterize such transport. The apparent permeabilities indicate that the methylated species show low absorption, whereas As(V) is a compound with moderate absorption. The kinetic study shows only a saturable component for MMA(V) transport in the range of concentrations assayed. The existence of paracellular transport was shown for all of the species, with greater significance in the case of the methylated forms. As(V) absorption was inhibited by 10 mM phosphate, and a phosphate transporter therefore could take part in intestinal absorption. Acidification of the medium (pH 5.5) resulted in a marked increase in As(V) and DMA(V) permeability (4-8 times, respectively) but not in MMA(V) permeability. This makes it necessary to consider the possible existence of absorption in the proximal intestine and even in the stomach, where the environment is acidic; alternatively, an H(+)-dependent transporter may be involved. The results obtained constitute the basis for future research on the mechanisms involved in the intestinal absorption of arsenic and its species, a decisive step in relation to their toxic action.

In vitro study of intestinal transport of inorganic and methylated arsenic species by Caco-2/HT29-MTX cocultures.

This study characterizes intestinal absorption of arsenic species using in vitro system Caco-2/HT29-MTX cocultures in various proportions (100/0 to 30/70). The species assayed were As(V), As(III), monomethylarsonic acid [MMA(V)], monomethylarsonous acid [MMA(III)], dimethylarsinic acid [DMA(V)], and dimethylarsinous acid [DMA(III)]. The results show that the apparent permeability (P(app)) values of pentavalent species increase significantly in the Caco-2/HT29-MTX cocultures in comparison with the Caco-2 monoculture, probably because of enhancement of paracellular transport. For MMA(III) and DMA(III), P(app) decreases in the Caco-2/HT29-MTX cell model, and for As(III), there is no change in P(app) between the two culture models. Transport studies of arsenic solubilized from cooked foods (rice, garlic, and seaweed) after applying an in vitro gastrointestinal digestion showed that arsenic absorption also varies with the model used, increasing with the incorporation of HT29-MTX in the culture. These results show the importance of choosing a suitable in vitro model when evaluating intestinal arsenic absorption processes.

Commonalities in Metabolism of Arsenicals

Environmental Context. Health effects associated with inorganic arsenic include various cancers and increased risk of diabetes. Millions of people in Bangladesh and India are at risk through use of contaminated drinking water. When humans ingest inorganic arsenic, it is rapidly converted to methylated metabolites. Although this methylation process is largely understood, the metabolism of other arsenicals (e.g. arsenosugars to dimethylarsenic) is very unclear. Connections among pathways for metabolism of various arsenicals are now being elucidated. Commonalities and differences in these pathways may be important determinants of the risk associated with exposure to these agents. Abstract. Elucidating the pathway of inorganic arsenic metabolism shows that some of methylated arsenicals formed as intermediates and products are reactive and toxic species. Hence, methylated arsenicals likely mediate at least some of the toxic and carcinogenic effects associated with exposure to arsenic. Trimethylarsonium compounds and arsenosugars are two other classes of arsenicals to which humans are routinely exposed and there is evidence that both classes are metabolized to produce methylated arsenicals. Here, we review evidence for production of methylated metabolism and consider the challenges posed in unraveling a complex web for metabolism of arsenicals in humans.

Metabolism of inorganic arsenic in intestinal epithelial cell lines.

This study evaluates the metabolism of inorganic arsenic (iAs) [As(III) and As(V)] in human intestinal cells as a function of cell type, differentiation stage, type of support used for cell growth, and exposure time. Additionally, mRNA expression of arsenic (+3 oxidation state) methyltransferase (AS3MT) was evaluated. For this purpose, Caco-2 (absorptive type) and HT29-MTX (goblet type) cells were exposed at various stages of differentiation (5, 15, and 21 days post-seeding) with different concentrations of As(III) and As(V) (1 and 10 μM) and exposure times (24, 48, and 72 h), using multiwell plates or Transwells. The results show that both cell lines express AS3MT at all stages of differentiation and in all culture conditions. Caco-2 cells are capable of metabolizing iAs, As(III) metabolism being greater than that observed for As(V). Metabolism depends on the stage of differentiation, reaching 36% after 48 h of exposure of differentiated cells (15 days post-seeding), with the monomethylated species as the major metabolite. Analysis of the cell interior shows that the metabolites are present predominantly in trivalent form. The type of support is also an important factor, metabolism being greater in multiwell plates than in Transwells (36 ± 6% vs 11 ± 3%). Neither monomethylated arsenic species (MMA) nor dimethylated arsenic species (DMA) are detected in HT29-MTX cells after exposure to iAs, possibly because most of the iAs is retained in the mucus layer and does not internalize. These results show that the intestine is an organ that may take part in presystemic metabolism of iAs. Moreover, the transformation of iAs into more toxic species indicates the need to study the effects of this species on the intestinal epithelium.

Quantification of fluoride in food by microwave acid digestion and fluoride ion-selective electrode.

To quantify fluoride in food it is necessary to extract the fluoride from the matrix. Dry ashing (alkali fusion) and facilitated diffusion are the methods most commonly used, but their application requires lengthy treatments. The present study proposes the use of a microwave oven and 7 mol/L nitric acid for simple, rapid digestion of foods for fluoride analysis. The analyte is subsequently quantified by fluoride ion-selective electrode. The various steps of the method were optimized and an in-house validation was performed. The limit of quantification (0.130 mg/kg), trueness (92%), recovery (84-101%), and precision (1-8%) were determined. These analytical characteristics are satisfactory and show the suitability of the method for analysis of fluoride in foods of various kinds. The methods ease of application and the use of equipment normally found in food analysis laboratories may help to further increase research on fluoride concentrations in foods consumed by the population.

Dietary Strategies To Reduce the Bioaccessibility of Arsenic from Food Matrices

The main route of exposure to arsenic (As) is the consumption of water and foods, in which the forms with greatest toxicity are inorganic As and dimethylarsinic acid, DMA(V). The objective of this study was to search for dietary components that reduce the bioaccessibility of As from food and water, in order to reduce the amount of As available for absorption. For this purpose, 35 compounds were assayed by use of a static in vitro model of gastrointestinal digestion. Sulfates of Fe(II) and Fe(III) reduced the solubility of inorganic As (86-99%) and DMA(V) in aqueous solution (40-66%). This reduction was also observed in rice (100%) and seaweed (60%). Aluminum, titanium, and tannic acid also reduced the bioaccessibility of As from food (42-70%). These data show that the use of dietary components may be a good strategy to reduce the entry of As into systemic circulation.